The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein or peptide at a specific site with a functional group of orthogonal reactivity which can in turn be used for protein modification or immobilization.
Site-specific derivatization of proteins and peptides is useful in a variety of research and therapeutic applications. For example, attachment of reporter molecules (labels or tags) can be used to detect the proteins or peptides. Attachment of ligands which bind to a receptor or other binding partner can, for example, be used to facilitate protein or peptide detection, isolation or purification. Linking of therapeutic proteins to other proteins or peptides, or to ligands or other small molecules, for example, can enhance their therapeutic value.
Site-specific derivatization of proteins and peptides is also useful for their immobilization onto surfaces. Microarrays in which proteins, peptides or other chemical species are immobilized to a surface enable high-throughput experiments that require only small amounts of analyte. For example, protein “chips” can be used to detect protein-ligand, protein-protein, and antibody-antigen interaction. Attaching proteins or other chemical species covalently, rather than non-covalently produces more robust surfaces. Methods and reagents for immobilization that result in the formation of covalent and stable linkages provide significant benefit. Additionally, the ability to attach proteins or other chemical species to a surface in a uniform rather than random manner and which results in high-density attachment can provide a substantial advantage in assay sensitivity.
Protein microarrays [53, 54] facilitate high-throughput approaches for the discovery and characterization of protein-ligand interactions which are useful in the study of complicated biological pathways. Additionally, protein microarrays facilitate high-throughput methods of screening small molecule libraries as potential enzyme inhibitors and protein binding agents which are important in drug development. Moreover, protein microarrays provide recyclable devices that can be used to detect disease biomarkers in fluids of patients and are a great aid in disease diagnosis.
While DNA microarrays [55] have been produced in a large scale to study gene function, the fabrication of protein microarrays is more arduous. A major complicating factor is the tremendous chemical complexity of proteins. Proteins consist of 20 amino acids, each bearing a different side chain with distinct chemical properties, some of which possess further diversity as a result of post-translational modification. Moreover, the same amino acid exhibits different reactivity when present in different positions in a protein. In addition, proteins have limited stability and are susceptible to loss of activity when subjected to chemical modification. Accordingly, one of the major challenges in protein microarray technology is the development of general and facile strategies for protein immobilization.
Proteins have been immobilized on surfaces both non-covalently and covalently [56]. Facile, non-covalent immobilization has been achieved by physical adsorption [31, 57] and affinity tag mediated complex formation reactions [58, 59]. Nonetheless, covalent immobilization of proteins results in a more robust protein array.
Proteins are nucleophilic. Their side chains contain no electrophiles other than the disulfide bonds of cysteines or functional groups installed by post-translational modification. Accordingly, the chemical reactivity of proteins necessarily entails nucleophilic side chains, such as those of lysine [1] and cysteine [2,3]. The prevalence of these residues obviates control over the regiochemistry of reactions [4], producing heterogeneous reaction products often at the expense of biological function [3,5].
Covalent immobilization via nucleophilic side chains of lysine and cysteine residues [1, 60] produces a randomly oriented protein array. In contrast, site-specific covalent immobilization, either via an engineered cysteine [3] or via a non-proteinogenic group installed in a protein, affords a uniformly oriented protein array. Oriented protein arrays exhibit higher ligand binding ability [3, 61] and better reproducibility of enzymatic activity [62] when compared to arrays generated by random immobilization.
An intermediate that forms during the intein-mediated splicing of proteins contains an electrophile—a thioester (FIG. 1) [6]. The orthogonal reactivity of this functional group (as an electrophile) compared to the predominantly nucleophilic groups of proteins an be exploited for the site-specific modification of a protein by reaction with cysteine derivatives [7] or tandem reaction with a small-molecule thiol and amine [8]. Although thiols are potent nucleophiles for thioesters, the resultant thioesters are inherently unstable to hydrolysis [9], making the simple transthioesterification of an intein-derived thioester unsuitable for the chemical modification of proteins.
The powerful methods of native chemical ligation [10] and expressed protein ligation [11] offer an ingenious solution to this problem. After transthioesterification with a cysteine residue, S→N acyl transfer regenerates the thiol and forms a stable amide linkage. This approach, which has been used for protein modification and immobilization [12,13], however introduces a residual thiol that can be the focal point for undesirable side reactions. For example, cysteine is by far the most reactive residue toward disulfide bonds, O2(g), and other common electrophiles [14]. In addition, the sulfhydryl group of cysteine can undergo β-elimination to generate dehydroalanine [15], or disrupt self-assembled monolayers on gold or silver surfaces [16]. An alternative means to exploit intein-derived thioesters for the installation of an orthogonal functional group is needed in view of these detrimental attributes.
In contrast to sulfur nucleophiles, nitrogen nucleophiles could, in theory, react directly with the thioesters formed during intein-mediated protein splicing to form inert linkages. This reaction has been neither explored nor exploited previously. Additionally, reagents carrying a nitrogen nucleophile and a functional group exhibiting orthogonal reactivity (a hetero bifunctional reagent) could, again in theory, both attack an intein-derived thioester to form a stable linkage and install the orthogonal (and thus useful) functional group.
The azido group can serve in many applications as an orthogonal functional group, being absent from natural proteins, nucleic acids, and carbohydrates [17]. Moreover, chemical reactions of the azido group, such as the Cu(I)-catalyzed Huisgen 1,3-dipolar azide-alkyne cycloaddition [18] and Staudinger ligation [19] could be used for site-specific modification or immobilization. Azido-proteins have been produced previously. For example, Schultz and coworkers have developed a method for incorporating azidolysine into proteins [20]. Their approach involves producing a suppressor tRNA charged with azidolysine that inserts that residue into a protein as directed by an engineered gene. This method, although site-specific, is labor intensive and low yielding. Tirrell, Bertozzi, and coworkers have incorporated an azido group into a protein by using azidohomoalanine, which replaces methionine in proteins produced in methionine-depleted bacterial cultures [21]. This method is not site-specific for proteins containing more than one methionine residue.
U.S. Pat. No. 6,972,320 reports the coupling of peptides and proteins derivatized with azido groups with those derivatized with phosphinothioester groups via reaction of these functional groups to form an amide bond. U.S. patent application 2005/0048192 (Raines and Soellner) published Mar. 3, 2005 reports the use of azido functional groups for site-selective rapid and high-yielding covalent ligation of molecules, including peptides and proteins, to surfaces. The ligation is based on the reaction of an azide with a phosphinothioester to form an amide bond through which the molecule is immobilized on the surface.
Methods and reagents that would allow derivatization at thioesters formed in proteins, particularly those formed during intein-mediated splicing, where the resulting linkage is stable and which further allow incorporation of a functional group with orthogonal reactivity, such as an azide, for subsequent reaction would clearly be useful in the art.